Characterization of a monoclonal rat anti-mouse interleukin 2 (IL-2) receptor antibody and its use in the biochemical characterization of the murine IL-2 receptor (original) (raw)
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A NEW ANTI-RAT INTERLEUKIN-2 RECEPTOR ANTIBODY AND A RECEPTOR ASSAY
A mouse anti-rat interleukin-2 receptor (IL-2R) monoclonal antibody (mAb), ART-65, has recently been developed which recognizes the rat IL-2R but binds to an epitope distinct from that recognized by the mAb ART-18. The availability of two mAb against the rat IL-2R molecule permitted the development of an enzyme-linked immunosorbent assay (ELISA) which detects soluble and solubilized rat IL-2R. Using this ELISA, time course studies of ConA and LPS-activated rat splenocytes revealed that IL-2R are released into their environment during the culture period, and that IL-2R can be detected in rat sera. The significance of the results is discussed.
Journal of Immunological Methods, 1987
A monoclonal antibody prepared against the murine interleukin-2 receptor (IL-2R) was employed to develop an ELISA method for measuring the immunological activation of T-cells. The assay detects an increase in IL-2R expression on activated lymphocytes. Stimulated splenic lymphocytes displayed markedly higher IL-2R expression compared to unstimulated controls. A significant increase in IL-2R expression on lymphocytes was detected in mitogen-stimulated responses, in a one-way mixed leukocyte reaction (MLR) and in the antigen-specific responses to conalbumin and purified protein derivative (PPD) in vitro. At a constant cell number, the level of IL-2R expression was found to be dependent on the dose of the stimulant. A comparative study of the kinetics of activation of splenic lymphocytes in response to mitogen, antigen and allogeneic cells as measured by the IL-2R ELISA and the conventional tritiated thymidine (3HTdR) uptake assay revealed remarkable similarity. For both assays, the mitogenic response was detected within 12 h and peaked at 72 h, the MLR was detectable within 2-3 days and peaked at day 6, and the specific antigenic response was detected within 2 days and peaked on day 4-5. Hydroxyurea, an inhibitor of DNA synthesis, had no effect on early IL-2R expression by mitogen-stimulated splenic lymphocytes, however, only 20% of maximum IL-2R expression could be detected at later stages of incubation. In contrast, cycloheximide, an inhibitor of protein synthesis, completely abrogated IL-2R expression and proliferation of stimulated lymphocytes.
Activation of rat T lymphocytes by anti-CD2 monoclonal antibodies
The Journal of …, 1988
The CD2 antigen (Tll, E-rosette receptor) was first identified on human T lymphocytes with mAbs (1-3). The antigen is a glycoprotein of 50,000 apparent M, and recently the protein sequences for CD2 of human (4, 5), rat (6), and mouse have been derived from cDNA sequences . In each case the sequence indicates two external domains that are Ig related, one transmembrane sequence, and a cytoplasmic domain with 115-116 amino acids .
An ELISA that detects cells-associated and released rat IL-2 receptors in a soluble form
1987
A mouse anti-rat interleukin-2 receptor (IL-2R) monoclonal antibody (mAb), ART-65, has recently been developed which recognizes the rat IL-2R but binds to an epitope distinct from that recognized by the mAb ART-18. The availability of two mAb against the rat IL-2R molecule permitted the development of an enzyme-linked immunosorbent assay (ELISA) which detects soluble and solubilized rat IL-2R. Using this ELISA, time course studies of ConA and LPS-activated rat splenocytes revealed that IL-2R are released into their environment during the culture period, and that IL-2R can be detected in rat sera. The significance of the results is discussed.
A study on OX39, a murine anti-rat interleukin 2 receptor antibody
Transplant International, 1988
OX39, a murine IgG1 monoclonal antibody (MoAb) that recognizes the 55 kDa alpha chain of the rat interleukin 2 receptor (R-IL2), was studied in vitro for its ability to interfere with IL2 binding and IL2-induced proliferation on rat concanavalin A (ConA) blasts and in vivo in a model of rat heart allografts. In vitro studies indicated that OX39 MoAb interacts with a single class of sites on the alpha chain of the rat R-IL2 with a high affinity (KD=0.8 nm) and competes with IL2 binding on this chain (KI = 0.53 rim). In contrast, OX39 MoAb was found to be 10-20 times less efficient in competing with IL2 binding to the high-affinity R-IL2 (KI~a0 nm). It is proposed that the epitope recognized by OX39 on the alpha chain (low-affinity R-IL2) is modified on (or buried in) the high-affinity R-IL2 configuration. Accordingly, OX39 was found to be a weak inhibitor in vitro on IL2-induced proliferation and in vivo on allograft rejection. Allograft survival was unaffected by doses of OX39 of 20 and 50 ~tg/rat for 9 days; only a borderline effect was noted when doses as high as 250 t, tg/rat were used. A significant, but restricted, effect of OX39 could be further detected when combined with low doses of cyclosporine A (1.5 mg/kg), which were ineffective by themselves. Together, our data suggest that in order to be efficient in vivo, anti-R-IL2 MoAbs must bind with high affinity to epitopes involved in the high-affinity IL2 binding site.
Characterization of the interleukin 2 receptor beta chain using three distinct monoclonal antibodies
Proceedings of the National Academy of Sciences, 1989
The human high-affmity receptor for interleukin 2 (IL-2) has been proposed as being a membrane complex composed of at least two distinct polypeptide chains: p55 (a chain), recognized by the anti-Tac monoclonal antibody (mAb), and p75 ((chain), both of which are capable of binding IL-2. Whereas the a chain itself has been shown to be nonfunctional, the (3 chain appears to be pivotal in the IL-2 signal transduction, although the (3 chain is otherwise poorly characterized. Three (3 chain-specific mAbs, designated Mik-(31,-(32, and-(33, were developed. Mik-(81 and-132 completely inhibited the IL-2 binding to the P chain, whereas Mik-83 immunoprecipitated the (3 chain crosslinked with 125I-labeled IL-2. The (3 chain immunoprecipitated by these mAbs was revealed to have a Mr of 68,000-72,000. High-affimity IL-2 binding was completely abolished by Mik-(31. Although IL-2-dependent T-cell growth at high IL-2 concentrations was not inhibited by the anti-Tac, it was almost completely inhibited by Mik-(81 in the presence of the anti-Tac. These results clearly indicate that the (3 chain is an indispensable component to the high-afflinity IL-2 receptor and is responsible for the IL-2 signal transduction. The (3 chain was found to be constitutively expressed without the a chain on the surface of peripheral blood Leu-19+ natural killer cells. The growth of T cells is regulated by interaction between interleukin 2 (IL-2) and its receptor (IL-2R) (1, 2). Recent chemical crosslinking studies (3-5) using 1251I-labeled IL-2 (125I-IL-2) have suggested that the high-affinity receptor for IL-2 having a dissociation constant (Kd) of =10 pM is a membrane complex composed of at least two distinct polypeptide chains; one is the Mr 55,000 peptide (p55 or a chain) recognized by the monoclonal antibody (mAb) anti-Tac (6, 7), and the other is a peptide with a putative Mr of 75,000 (p75 or 1 chain). Unlike many multisubunit receptors, each of the two subunits ofthe IL-2R can be individually expressed in the absence of the other. Moreover, individually existing a and p chains are capable of binding IL-2 with low (Kd 10 nM) and intermediate (Kd 1 nM) affinities, respectively (8, 9).
Journal of Leukocyte Biology, 1990
Foreign antigens or mitogens initiate activation of T lymphocytes through antigen-specific receptors. After antigen-receptor interaction, T cells release a lymphotrophic hormone, Interleukin 2 (IL2), and high-affinity IL2 receptors (IL2R) are progressively expressed on their surfaces. When evaluated under the scanning electron microscope after specific immunolabeling with colloidal gold markers, the density of expression of the 55 kd low-affinity IL2 receptor (Tac) increases in a time-dependent manner, reaching a maximum 72 hours after the onset of phytohemagglutinin (PHA) activation. In contrast, the number of cells expressing the 55 kd molecule reaches a maximum by 24 hours and remains constant through 72 hours. Immunogold labeling allows simultaneous determination of IL2 receptor density at the level of each individual cell and of the percentage of IL2 receptor-positive cells in any cell population under study. The rank order of receptor expression on the surface of established c...